Hepatitis E virus, known previously to cause much acute viral hepatitis in developing countries, has recently been identified as a cause of sporadic hepatitis in industrialized countries. It also has been found to cause chronic hepatitis in severely immune-compromised persons such as those receiving organ transplants. Inability to propagate the virus efficiently in cell culture or in small animal models has inhibited attempts to characterize its molecular biology or identify its pathogenic determinants. Two genotypes of the virus infect humans only, but the remaining two genotypes that infect humans are zoonotic and swine are a known reservoir. It seems certain that other animal reservoirs for human HEV must exist but they have yet to be identified and the biological factors that permit some strains to cross species boundaries are completely unknown. In FY2010, we isolated a new strain of genotype 3 HEV from the stool of a chronically infected patient and serially passaged this new virus strain in cultured human hepatoma cells and adapted it to grow relatively efficiently in these cells. During the adaptation process we discovered that a recombinant HEV with a 174 nucleotide insertion as well as numerous point mutations was selected.We made an infectious cDNA clone of this virus. Ribavirin has been used to treat a small number of patients chronically-infected with genotype 3 HEV. In FY2014, we continued testing genotypes 1, 2, and 3 viruses for sensitivity to ribavirin in cell-culture. In collaboration with Dr. Neyts in Belgium, we created a luciferase-expressing genotype 3 virus and used it to study hepatitis E virus sensitivity to ribavirin and to interferon. We found that ribavirin inhibited hepatitis E virus replication through depletion of cellular GTP pools. In collaboration with Dr. Girones in FY2014, we showed that the inactivation kinetics of HEV and human adenovirus 2 are equivalent indicating that chlorine inactivation is an effective strategy to control HEV waterborne transmission. in collaboration with Dr. Purcell in FY2014, we analyzed sera from transfused patients before screening to determine the incidence of hepatitis E transmission.